Studies on Enzymes of Prodigiosin Biosynthesis

Prodigiosin is a natural red tripyrrolic pigment with potent anti-cancer properties1. In a previous BBSRC-funded collaboration with Prof. George Salmond (Biochemistry), we elucidated the pathway for its biosynthesis and the enzymes responsible. One branch of the pathway makes a monopyrrole MAP and the steps are catalysed by the enzymes PigD, PigE, and PigB in that order. In this project we propose to study these three over-expressed and purified enzymes in vitro to verify that they do indeed catalyse the reactions proposed for them. PigD is proposed to be a TPP-dependent enzyme that transfers an acetyl group from pyruvate onto the B-position of an octenoyl derivative, probably a thioester, but the natural substrate has not been established. PigE seems to be a bifunctional enzyme that both reduces the thioester to an aldehyde and then performs a PLP-dependent transamination of the aldehyde. However the C-terminal half, proposed to be the reductase domain, is unlike any known reductase, so it will be particularly interesting to find out whether and how it catalyses the reduction. As well as studying its catalytic activity, we will try to obtain a crystal structure of PigE, not only because it is an entirely novel enzyme but also because their is an intriguing possibility that the reactive aldehyde intermediate gets channelled direct from one active site to the other without being released into solution. PigB is proposed to be a flavin-dependent oxidase that oxidises a dihydropyrrole to the pyrrole. We have previously synthesised the dihydropyrrole (and shown that it does restore prodigiosin biosynthesis to a mutant blocked earlier in the pathway2), however we were not able to effect the oxidation to a pyrrole by purely chemical means. Therefore PigB may be a useful (and cheap as it only uses O2) way to effect this valuable transformation. After establishing its activity in vitro we will study a range of substrate analogues to find out how broad a range substrates it can accept. We will also investigate whether the living bacterium can use these substrate analogues to make new analogues of prodigiosin.
This project will be an equal collaboration between Prof. Salmond and myself and requires skills from both groups, particularly molecular biology, genetic engineering and protein expression in Prof. Salmond's lab and synthesis and enzyme kinetics in my lab. It is therefore interdisciplinary. Prodigiosin's anti-cancer properties (not to mention the numerous other biological activities that have been reported) make it an important target. A close analogue of prodigiosin (obatoclax) has been put through Phase 2 clinical trials for treatment of several different types of cancer. Therefore the ability to make large quantities of either natural prodigiosin or closely related compounds by fermentation may be very valuable. To achieve this goal it is important that the activities of each enzyme are well understood. Therefore this study may contribute to both Industrial Biotechnology and Bioscience for Health.